Bottom Line:
The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers.The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC.Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

ABSTRACTAIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2(PYD)) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

f6: AIM2FL assembles into filaments without dsDNA.(a) A negatively stained electron micrograph of AIM2FL at 2 μM. (b) A negatively stained electron micrograph of AIM2Hin at 5 μM. (c) Higher magnification of the AIM2 filament. The inset is unpicked Brussels sprout, and the cartoon on the right is the proposed overall arrangement of the filament. The red boxes in c and d indicate the stable ‘core stem' of the AIM2FL filament. (d) An electron micrograph of AIM2FL–eGFP (2 μM). The cartoon on the right is the proposed overall arrangement of the filament. (e) Electron micrographs of AIM2FL mutants at 2 μM. Scale bar, 100 nm.

Mentions:
The autoinhibitory model entails that dsDNA is required to unlock monomeric AIM2 and initiate oligomerization3233. By contrast, because untagged AIM2FL binds dsDNA much more tightly than AIM2Hin, we hypothesized that the role of dsDNA is to simply increase the local concentration of AIM2 by acting as a ‘one-dimensional ruler,' consequently improving the prospects for forming AIM2PYD·AIM2PYD encounter complexes. This new model entails that dsDNA-free AIM2FL should be able to oligomerize on its own if the concentration threshold is met. On the other hand, apo-AIM2FL should remain monomeric according to the autoinhibitory model. To test these opposing predictions, we used negative stain electron microscopy (ns-EM) to probe the oligomeric state of apo-AIM2FL at various concentrations. Disagreeing with the autoinhibitory model, we found that apo-AIM2FL forms filaments in a protein concentration-dependent manner (≥1 μM; Fig. 6a, Supplementary Fig. 2a). By contrast, we did not observe any auto-assembled AIM2Hin filaments (Fig. 6b), and MBP-AIM2FL showed aggregates clearly different from the filamentous structures observed from untagged AIM2FL (Supplementary Fig. 2b). AIM2FL filaments were detected even at 1.6 M NaCl, thus suggesting that they are more resilient against the environment than the ASCPYD filament7 (Supplementary Fig. 2c). Considering the auto-oligomerization activity, we kept the concentration of wild-type AIM2FL as low as possible in our biochemical assays (typically <100 nM; Figs 2, 3, 4, 5). Moreover, we observed saturating binding isotherms in most of our assays (Fig. 2a), suggesting that auto-oligomerization did not cause any significant artifacts.

f6: AIM2FL assembles into filaments without dsDNA.(a) A negatively stained electron micrograph of AIM2FL at 2 μM. (b) A negatively stained electron micrograph of AIM2Hin at 5 μM. (c) Higher magnification of the AIM2 filament. The inset is unpicked Brussels sprout, and the cartoon on the right is the proposed overall arrangement of the filament. The red boxes in c and d indicate the stable ‘core stem' of the AIM2FL filament. (d) An electron micrograph of AIM2FL–eGFP (2 μM). The cartoon on the right is the proposed overall arrangement of the filament. (e) Electron micrographs of AIM2FL mutants at 2 μM. Scale bar, 100 nm.

Mentions:
The autoinhibitory model entails that dsDNA is required to unlock monomeric AIM2 and initiate oligomerization3233. By contrast, because untagged AIM2FL binds dsDNA much more tightly than AIM2Hin, we hypothesized that the role of dsDNA is to simply increase the local concentration of AIM2 by acting as a ‘one-dimensional ruler,' consequently improving the prospects for forming AIM2PYD·AIM2PYD encounter complexes. This new model entails that dsDNA-free AIM2FL should be able to oligomerize on its own if the concentration threshold is met. On the other hand, apo-AIM2FL should remain monomeric according to the autoinhibitory model. To test these opposing predictions, we used negative stain electron microscopy (ns-EM) to probe the oligomeric state of apo-AIM2FL at various concentrations. Disagreeing with the autoinhibitory model, we found that apo-AIM2FL forms filaments in a protein concentration-dependent manner (≥1 μM; Fig. 6a, Supplementary Fig. 2a). By contrast, we did not observe any auto-assembled AIM2Hin filaments (Fig. 6b), and MBP-AIM2FL showed aggregates clearly different from the filamentous structures observed from untagged AIM2FL (Supplementary Fig. 2b). AIM2FL filaments were detected even at 1.6 M NaCl, thus suggesting that they are more resilient against the environment than the ASCPYD filament7 (Supplementary Fig. 2c). Considering the auto-oligomerization activity, we kept the concentration of wild-type AIM2FL as low as possible in our biochemical assays (typically <100 nM; Figs 2, 3, 4, 5). Moreover, we observed saturating binding isotherms in most of our assays (Fig. 2a), suggesting that auto-oligomerization did not cause any significant artifacts.

Bottom Line:
The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers.The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC.Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

ABSTRACTAIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2(PYD)) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.